Cytopathology
Palpation guided fine needle aspiration
Smear making and special techniques for recovering and dividing aspirate material

Author: Joe D. Jakowski, M.D. (see Authors page)

Revised: 8 February 2018, last major update August 2013

Copyright: (c) 2002-2018, PathologyOutlines.com, Inc.

PubMed Search: Smear preparation [title]

Cite this page: Jakowski, J. D. Smear making and special techniques for recovering and dividing aspirate material. PathologyOutlines.com website. http://www.pathologyoutlines.com/topic/cytopathologypgfnasmearmaking.html. Accessed December 17th, 2018.
Overview
  • Special studies may be applied to the aspirate material obtained from FNA but most aspirators just spread a portion of FNA material on a glass slide for examination by routine light microscopy
  • Preparation of aspirate smears is by far the least expensive and most rapid type of preparation of FNA material for diagnosis
  • IMPORTANT:
    1. The preparation of high quality smears is one of the most important parts of the entire FNA procedure
    2. Although the smearing techniques may seem tedious, they can be mastered with practice and executed within a few seconds
  • High quality smears:
    • Are prepared by methods tailored to physical properties of FNA material, for example cyst fluid versus semisolid material
    • Cells are spread out in a monolayer and stains so they easily transmit light
    • Specimen is concentrated enough in a small portion of slide for ease of screening and microscopic review
    • Cells are well preserved without crush artifact
    • Any semisolid tissue fragments are spread out with gentle pressure with some remaining intact
    • Cellular material is not excessively trapped in blood clot
    • Slides are properly labeled with at least two patient identifiers
  • Several stains and special methods may be applied to the material on aspirate smear slides:
    • Air dry and stain with rapid Romanowsky type stains (e.g. Diff-Quik)
    • Immediate fixation (e.g. 95% ethanol) or rehydration of air dried smears for H&E or Papanicolaou staining
    • Histochemical staining (e.g. AFB, Congo Red, GMS)
    • Immunocytochemistry
    • Molecular testing (e.g. FISH, mutational analysis such as KRAS, EGFR)
Type of FNA material obtained for smear making
  • Three general categories of physical properties of FNA material which suggest how to triage the material and what smear making methods to use:

Solids:
  • Distinct firm fragments of visible material that appear in background of other fluid or semisolid material or are expressed out as a single "core" or as fragments in a clean background
  • Often difficult or impossible to smear as a monolayer
  • May roll about the slide, leave "streak marks" or be extensively crushed if smearing is attempted
  • Often create air bubbles under cover slips as they are very thick
  • Examples: tissue fragments from very firm sarcomas, fibrous / scar tissue or even normal skeletal muscle, bone, cartilage, foreign body or calcified material

IMPORTANT: This firm solid material may be best triaged by picking it off the slide and submitting it for cell block or perhaps performing a touch preparation or a squash preparation instead of attempting to make a smear

Semisolid and special types of semisolids:
  • Most common aspirate material obtained, consisting of visible minute soft particulate material often in a slightly bloody background
  • Tends to be moderate to highly cellular material and often yields good material for diagnosis unless necrotic
  • Particulate material tends to spread easily about the slide when smeared
  • May cause "thick smear" with difficult interpretation if too much material placed on slide for smearing or if concentrated too much in a limited area
  • Creates low to moderate hydrostatic forces between smearing slides
  • Examples: tumor and tumor like conditions including solid thyroid nodule, lymph node, breast tumor, abscess, epidermal inclusion cyst
  • Adipose tissue and fatty / lipid containing material:
    • Often accompanied by oily droplets on smear slide
    • Does not air dry well and stays "greasy"
    • Is hydrophobic, and does not stay on slide very well after staining
    • Examples: aspirates from fat containing tumors such as lipoma or well differentiated liposarcoma

Fluid:
  • Watery like material that does not "stick" to the slide very well
  • Tends to run all over the slide when slide is tilted even slightly
  • Usually has very low concentration of cells
  • Creates high hydrostatic forces between smearing slides
  • Examples: thin serous fluid from thyroid cysts, fibrocystic changes in breast, seroma
Transferring FNA material from needle onto glass slide
General:
  • FNA material must be rapidly transferred from needle onto glass slide to make good quality smears for diagnosis
  • Clotting of FNA material within needle, if transfer is delayed, may cause material to be trapped within needle or hub or smear to be excessively thick or clotted, causing a limited or obscurred microscopic evaluation

IMPORTANT: No smearing technique can correct clotting artifact

Techniques to ensure best results in transferring FNA material from needle to glass slide include:
  • Keep most of FNA material within needle shaft during aspiration and stop the aspiration when material is seen entering the hub
  • Perform aspiration in rapid manner (typically less than 5 - 10 seconds)
  • Set up FNA procedure so that material is expressed onto glass slide within seconds of needle removal from patient
  • Make sure needle is easily removed from syringe tip (especially with Leur lock tips) BEFORE aspiration occurs
  • Place glass slides for smear making as close as safely possible to aspiration site
  • Clear everything / everyone from path between aspiration site and slide making site as you will be moving rapidly between those areas with a "dirty needle"
  • Make smears immediately after you place material onto glass slide (seconds count!)
  • When expressing the material out of the needle using positive pressure from the syringe, keep the needle tip's beveled edge in contact with the glass slide surface using a 45 to 90 degree angle to the surface of the slide
  • Do not "spray" the FNA material over the glass slide so that material is broken up into tiny droplets about the slide, which will cause them to dry much faster than if a single small droplet is placed
Types of smear making methods
  • Several smear making methods and variations have been documented
  • Personal preference and teaching exposure influence the method of choice but the physical properties of the FNA material (including the concentration of hypocellular material or cyst fluid) can also dictate the most useful method of triaging and smear making

General:
  • Express the FNA material for smear making on the slide so that it is about 2/3 the way up from the non frosted end of the slide
  • Generally, one to two drops forming a single large droplet of semisolid FNA material should suffice per slide (1 drop = ~.05 mL)
  • For cyst fluid or liquid material, several drops up to 0.5 mL may be placed on the slide and concentrated using the special methods below
One step smear method
General:
  • Excellent for semisolid and small volume FNA material
  • Typically only one slide is produced that has smeared material for evaluation
  • Smear usually occupies less surface area than Two step pull method but this technique is more difficult than Two step pull method

Technique:
  • Hold up a slide with the droplet of FNA material (the "non spreader" slide); the drop should face you and the non frosted end should point at a steep angle towards the floor; use your non dominant hand by holding the frosted end pinched between your thumb and index finger and use the remaining fingertips of this hand to support the back of the slide
  • A second slide, called the "spreader slide", is then brought up with the dominant hand by grasping it by the frosted end pinched between the thumb, index and third finger to a position that is perpendicular above the "non spreader" slide
  • Keeping this perpendicular orientation, the middle of the "spreader slide" is then poised over the specimen droplet on the "non spreader" slide with its lower edge of the long axis touching the "non spreader" slide and forming a hinge like contact between them
  • "Spreader slide" is then rotated forward (while still maintaining the hinge like contact) onto the droplet with gentle pressure to flatten but not crush it
  • "Spreader slide" is then pulled down the surface of the "non spreader" slide (which is held steady) to smear the droplet using constant and gentle pressure on the "spreader slide" while continuing to maintain the perpendicular orientation of the two slides
  • The "non spreader" slide should have a good quality ovoid shaped smear
  • The "spreader slide" should have little to no material on it and may be discarded

Smear problems:
  • "Edge artifact": caused by uneven pressure on either edge of the "spreader slide" in which a single or multiple thick smear lines will be created; this results in an area that is difficult to evaluate due to excessive cell thickness; correct by holding the "smear slide" with even pressure throughout the smearing process so that the flat surfaces of the "smear slide" and "non smear slide" remain parallel
  • "Crush artifact": caused by too much pressure being placed on flat surfaces between the "spreader slide" and "non spreader" slide OR by too much hydrostatic forces between the slides (e.g. watery fluid); correct by decreasing the thumb pressure on "smear slide" and increase the speed of the smearing action; for cyst fluid, try using another smearing or concentration technique before smearing and also lessen the contact time between the initial lowering of the "smear slide" with the "non smear slide" and increasing the speed of the smearing action
Two step pull method
General:
  • Excellent for semisolid and small volume FNA material
  • Usually two slides are produced that have smeared material for evaluation
  • Usually produces a smear that occupies more surface area than the One step method
  • Technically less challenging than the One step method

Technique:
  • The slide with the droplet of FNA material is held up so it is essentially parallel with the floor by using the non dominant hand to hold the frosted end pinched between your thumb, index and third finger
  • A second slide is then brought up with the dominant hand in an inverted fashion by grasping it by the frosted end pinched between thumb, index and third finger
  • This inverted slide is then poised over the specimen droplet of the slide below in a parallel fashion with the edge of the non frosted end of the inverted slide touching just below the frosted end of the droplet slide forming a hinge like contact
  • The inverted slide is then lowered (while still maintaining the hinge like contact of the lower edge of the long axis) onto the droplet with gentle pressure to flatten but not to crush the droplet
  • Both slides are then pulled apart to smear the droplet while continuing to maintain the parallel arrangement of the slides using constant and gentle pressure
  • Both slide should have a good quality ovoid shape of smear material

Smear problems:
  • "Edge artifact": caused by uneven pressure on either non frosted edge of the slides when they are pulled apart in which a single or multiple thick smear lines will be created, results in an area that is difficult to evaluate due to excessive thickness; correct by holding both slides with even pressure throughout the smearing process so their flat surfaces remain parallel throughout the pulling process
  • "Crush artifact": caused by too much pressure being placed on the flat surfaces between the slides OR by too much hydrostatic forces between the slides (e.g. watery cyst fluid); correct by decreasing the pressure on one or both of the slides and increasing the speed of the smearing action; for cyst fluid try using another smearing or concentration technique before smearing and also lessen the contact time between the initial lowering of the inverted slide and increase the speed of the pulling action
Pop off method
General:
  • Rarely used technique (mostly of historical value only)
  • Does not involve smearing the FNA material
  • Produces a small thick and rounded button of FNA material for evaluation
  • Good at keeping "tissue like" and cellular fragments together and thus giving some semblance of a histologic tissue section
  • Two slides are produced that have material for evaluation
  • Technically easy to produce
  • Main disadvantage is that the button produced is composed of thick cells / tissue fragments that can make cytologic evaluation difficult

Technique:
  • The slide with a small droplet of FNA material is held up essentially parallel to the floor by using the non dominant hand by holding the frosted end pinched between your thumb and index finger and the remaining finger tips of this hand supporting the back of the slide
  • A second slide is then brought up with the dominant hand, grasping it by the frosted end pinched between the thumb, index and third finger and holding it in a perpendicular orientation over the bottom slide containing the droplet
  • Keeping this perpendicular orientation, the middle of the second slide is then poised over the specimen droplet with its lower edge of the long axis touching the droplet slide and forming a hinge like contact between them
  • This second top slide is then quickly rotated forward (while still maintaining the hinge like contact) and onto the droplet on the bottom slide with gentle pressure to flatten but not to crush the droplet
  • The second top slide is then quickly rotated back off the flattened out droplet (while still maintaining the hinge like contact)
  • Both slides should then have an irregular rounded button of material for staining and microscopic examination
Simple wick concentration method
General:
  • Good for concentrating non bloody fluids and large volume FNA material
  • Technically very easy

Technique:
  • The slide with the expressed fluid droplet of FNA material is allowed to sit on the table for a few seconds in order to allow the cells to sediment
  • Leaving the slide stationary on the table, a corner of a piece of gauze is then placed very minimally into one edge of the fluid droplet and the fluid is allowed to wick into the gauze
  • After a few seconds the gauze is removed and discarded
  • The concentrated material may then be smeared using the One step or the Two step pull method
Simple incline concentration method
General:
  • Good for concentrating non bloody fluids and large volume FNA material
  • Technically very easy

Technique:
  • The slide with the expressed fluid droplet of FNA material is allowed to sit on the table for a few seconds in order to allow the cells to sediment
  • The slide is then picked up slowly by holding it parallel to the floor by using the non dominant hand with the frosted end pinched between your thumb, index and middle finger
  • Using your dominant hand, a piece of gauze is placed along the long axis of the slide with the fluid droplet
  • The slide with the fluid droplet is then gently rotated in the direction of the gauze and the fluid on the slide should flow and wick into the gauze
  • After a few seconds the gauze is removed and discarded
  • The slide with the concentrated material may then be smeared using the One step or the Two step pull method
Two step method for concentration
General:
  • Good for concentrating fluids and large volume FNA material essentially using an a combination of surface tension and an incline type method of specimen concentration
  • Technically more challenging than simple incline concentration methods, but may concentrate the FNA material within a smaller area on the slide for ease of screening and examination

Technique:
  • A slide with the droplet of FNA material (the "non spreader" slide) is held up (drop facing you and with the non frosted end pointed at small angle down towards the floor) by using your non dominant hand by holding the frosted end pinched between your thumb and index finger and with the remaining finger tips of this hand supporting the back of the slide
  • A second slide, called the "spreader slide", is then brought up with the dominant hand by grasping it by the upper edge of the long axis near the frosted end by pinching between the thumb, index and third finger to a position that is parallel to the "non spreader" slide with the frosted ends facing each other
  • Keeping this parallel orientation, the non frosted end of the "spreader slide" is used to form a 45 degree, hinge like contact between the "non spreader" slide below the specimen droplet
  • "Spreader slide" is slid up into the FNA droplet pool (while still maintaining the hinge like contact) and paused until the material spreads along its edge
  • "Spreader slide" is then pushed down the surface of the "non spreader" slide (which is held steady) to about the middle lower end while keeping the parallel orientation of the two slides
  • The spreader slide is removed and what is left is a line of fluid material on the "non spreader slide"
  • The "non spreader slide" is then quickly inverted so that the fluid runs from the line of material towards the frosted end
  • The now concentrated line of material on the "non spreader slide" is then smeared up towards the non frosted end of the "non spreader slide" using the One step method
  • There will be some material left on the distal edge of the "spreader slide" that may be recovered by spreading on another clean slide using the Two step pull method
Modified two step method for concentration
General:
  • Good for concentrating fluids of large volume FNA material (up to 0.5 mL) using a wick and spreader slide method of concentration
  • Technically more challenging than simple wick concentration methods but may have the advantage of concentrating the FNA material within a smaller area on the slide for ease of screening

Technique:
  • The slide with the expressed fluid droplet of FNA material is allowed to sit on the table for a few seconds in order to allow the cells to sediment
  • The slide is then picked up slowly by holding it parallel to the floor by using the non dominant hand with the frosted end pinched between your thumb, index and middle finger
  • Using your dominant hand a piece of gauze is placed along the long axis of the slide with the fluid droplet
  • The slide with the fluid droplet is then gently rotated in the direction of the gauze and the fluid on the slide should flow and wick into the gauze
  • After a few seconds the gauze is removed and discarded
  • A second slide, called the "spreader slide", is then brought up with the dominant hand by grasping it at the frosted end by pinching between the thumb, index and third finger to a position that is parallel to the "non spreader" slide with the frosted ends facing each other
  • Keeping this parallel orientation, the non frosted end of the "spreader slide" is used to form a 45 degree, hinge like contact between the "non spreader" slide below the concentrated specimen
  • "Spreader slide" is then slid up through the concentrated specimen pool (while still maintaining the hinge like contact) towards the frosted end which will carry the material along its edge
  • The spreader slide is removed and what is left is a line of concentrated material on the "non spreader slide"
  • The concentrated line of material may then be smeared using the One step or the Two step pull method
  • Any material left on the distal edge of the "spreader slide" may be recovered by spreading on another clean slide using the Two step pull method
Method(s) for dividing a single smear into several smears
General:
  • Excellent for semisolid and large volume FNA material
  • Employed when too much FNA material is expressed onto a single slide and helps avoid making overly thick smears
  • Multiple slides may be produced that have smear material for evaluation

Technique #1:
  • Essentially a combination of the Pop off method followed by smear making using the One step or the Two step pull method
  • The slide with the droplet of FNA material is held up essentially parallel to the floor using the non dominant hand by holding the frosted end pinched between your thumb and index finger and with the remaining finger tips of this hand supporting the back of the slide
  • A second slide is then brought up with the dominant hand, grasping it by the frosted end pinched between the thumb and index finger and third finger, and holding it in a perpendicular position over the bottom slide containing the droplet
  • The middle of this second top slide is then poised over the specimen droplet with the lower edge of the long axis touching the bottom slide forming a hinge like contact
  • This second top slide is then quickly rotated forward (while still maintaining the hinge like contact) and onto the droplet on the bottom slide with gentle pressure to flatten but not to crush the droplet
  • The second top slide is then quickly rotated back off the flattened out droplet (while still maintaining the hinge like contact)
  • This second slide is then lifted entirely off the bottom slide
  • The bottom slide is quickly put down on the table and the free non dominant hand can pick up a clean slide and the second slide with the divided material is then smeared onto this clean slide using the One step or the Two step pull method
  • The bottom slide that was initially put down on the table can then be picked back up and smeared with another clean slide by using the One step or the Two step pull method; OR, if there is still enough material on this slide, can be divided again as above and additional smears created

Technique #2:
  • Simple and crude method of using the bottom edge of a clean spreader slide to split the FNA material
  • The slide with the droplet of FNA material ("non spreader slide") is held up and slightly tilted up along its long axis from parallel to the floor using the non dominant hand by pinching the frosted end between your thumb and index finger and with the remaining finger tips of this hand supporting the back of the slide
  • A second spreader slide is then brought up with the dominant hand, grasping it by the frosted end pinched between the thumb and index and third finger, and holding its non frosted edge in a perpendicular position over the droplet on the bottom slide
  • The spreader slide's flat edge is then lowered into a portion of the droplet on the bottom slide until it makes a 45 degree contact angle with the bottom slide
  • The spreader slide is then pulled upwards and off the long axis edge of the bottom slide
  • The spreader slide should now have a portion of the original droplet material on its bottom edge and this may be smeared onto another clean slide using the One step or the Two step pull method
  • The non spreader slide may then be smeared by using the One step or the Two step pull method; OR, partitioned again using this technique
Additional special techniques for recovering aspirate material
  • Ideally, the aspirate material obtained during FNA should be collected in the needle shaft and the aspiration stopped when material is seen entering the hub
  • At times, however, diagnostic material may be trapped in the hub that cannot be forced out by using positive pressure from a syringe or material may have inadvertently entered the tip or barrel of the syringe
  • Use these special collection methods to recover trapped material to make additional slide smears:

Needle snap technique: to recover material trapped in the hub of the needle
  • First, recover as much material as possible from needle shaft by forcefully blowing air through it with the syringe and onto the glass slide
  • Then secure the needle tip in a needle protection device (e.g. Point-Lok Device from SIMS Portex Inc)
  • Then pick up the needle protection device and position it just beyond the end of a new glass slide so the hub of the needle points at a small angle down and touches the glass slide surface about 1/3 of the way up from the non frosted end of the slide
  • Your free hand can grab the hub of the needle and pull it upward while the hand holding the needle tip protector remains steady which will result in bending of the shaft of the needle in a slight arch and the resulting tension will "load" the hub
  • The needle hub is then quickly released and it is allowed to "snap" rapidly and forcefully against the glass slide and this will expel any FNA material that was trapped in the hub onto the slide
  • The hub may then be rapidly grabbed again and "snapped" in a similar matter as above to remove any more material that may be trapped in the hub
  • The additional material obtained may now be quickly smeared using the one or two step smearing technique
  • Alternatively, you can secure the needle tip as above, then grab the hub and forcefully hit the open end of it against the surface of the slide to dislodge the material onto the slide

Syringe pop technique: to recover material trapped in the tip or barrel of the syringe by rapidly and forcefully blowing it onto the surface of the glass slide
  • The syringe without the needle is held in your hand as if making a fist with the tip pointing down and just above the surface of the glass slide
  • The plunger is pulled out gently with the free hand to fill the barrel with air
  • Using the palm of the free hand the plunger is then rapidly, forcefully and audibly pushed into the barrel which is being held steady so the tip remains just above the glass slide surface
  • CAUTION: if the tip of the syringe slams against the glass slide during this technique, the glass slide may shatter
  • The additional material that has been expelled from the syringe and onto the glass slide may now be quickly smeared using the one or two step smearing technique